Oncolytic Virus and Uses Thereof

Abstract

The present invention relates to an anti-cancer virus and uses thereof, specifically, to an anti-cancer virus in which the expression of the thymidine kinase (TK) gene is suppressed and which contains genes encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a complement regulatory protein; and uses of such an anti-cancer virus. The anticancer virus of the present invention maintains its efficacy even when injected intravenously, and can be used to treat various solid cancers and metastatic cancers in addition to superficial solid cancers. In addition, the anticancer virus of the present invention expresses a complement regulatory protein on the surface of the virus, thereby acquiring resistance to attacks by the internal complement system, and is stable in blood, and maintains stable anticancer activity when injected intravenously, thereby reducing the dosage of the virus and minimizing side effects of anticancer drugs. Therefore, the anticancer virus of the present invention can be usefully used for preventing or treating cancer.

Description

[Technical field]

[0001] The present invention relates to an anti-cancer virus and uses thereof, specifically, to an anti-cancer virus in which the expression of the thymidine kinase (TK) gene is suppressed and which contains genes encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a complement regulatory protein; and uses of such an anti-cancer virus. [Background technology]

[0002] Cancer is a major cause of death worldwide, and the enormous personal and social burden it places on people makes the development of innovative anti-cancer therapeutics increasingly important. Accordingly, various approaches based on advances in molecular biology are being attempted.

[0003] Oncolytic viruses, which are anti-cancer therapeutics that have been in the spotlight recently, are replicable and infectious viruses. They are used for cancer treatment by inserting specific genes that target genetically abnormal sites in tumor cells into wild-type or attenuated viruses. After viral replication, these genetically modified anti-cancer viruses selectively spread through tissues and cause cell lysis in cancer cells and blood vessels surrounding the cancer cells. A representative anti-cancer virus is Imlygic (talimogene laherparepvec) from Amgen, which was approved as a melanoma treatment by the Food and Drug Administration (FDA) in October 2015.

[0004] Meanwhile, intravenous injection of medicines is an easy way to quickly deliver drugs that cannot be administered orally to the whole body, and is one of the most preferred forms of administration due to its convenience. However, anticancer virus preparations have the limitation that they are quickly removed by the body's immune system when administered intravenously, reducing their efficacy. Therefore, anticancer virus preparations are generally injected directly into the target tumor rather than intravenously to increase the probability of the virus reaching the tumor.

[0005] The intratumoral injection method is generally effective for easily accessible superficial cancers such as melanoma, breast cancer, and head and neck cancer, but for carcinomas such as deep solid cancers, the administration of an effective dose is disadvantageous in that it depends on the technical ability of the doctor who performs the treatment. In addition, the intratumoral injection treatment process is very invasive, so it is not as easy to use as a repeated treatment method as intravenous injection.

[0006] Despite the advantages of intravenous injection, anti-cancer viruses are difficult to administer via intravenous injection because foreign substances (i.e., viruses) administered into the bloodstream are gradually removed by the body's defense mechanisms, reducing the activity of the anti-cancer viruses. In other words, when a virus travels through the body via the bloodstream, the body recognizes it as an enemy and activates the natural immune system to neutralize and remove it.

[0007] When the human body is exposed to bacteria or viruses that have invaded from the outside, the complement system, which is the first line of defense of the innate immune system, acts first. Complements are proteins present in the blood that surround the surface of foreign microorganisms and facilitate the phagocytosis of macrophages and neutrophils. Specifically, when a virus penetrates the blood, the proteins on the surface of the virus first bind to the complement, agglutinating with each other, activating the complement. The activated complement surrounds the surface of the virus, facilitating the phagocytosis of phagocytes (opsonization), and lyses the virus by opening holes in the surface of the virus. The remains of the lysed virus are then completely removed by the phagocytosis of macrophages.

[0008] In order to prevent damage to surrounding normal cells caused by excessive activation of the complement system, the human body expresses complement regulatory proteins such as CD55, CD46, and CD59 on the cell surface to regulate excessive complement activity. Among these, CD46 and CD59 proteins only suppress part of the complement activation pathway, while CD55 protein (Decay Acceleration Factor, DAF) is involved in all stages of complement activation and more strongly inhibits complement activation.

[0009] A portion of the anticancer viruses that grow in host cells (approximately 5-20%) are characterized by taking the form of "extracellular enveloped viruses (EEVs)" that are produced in a manner that is surrounded by the cell membrane of the infected cell. It is known that such EEVs avoid complement activation and have a long survival period in the blood, and the mechanism behind this is known to be due to complement regulatory proteins such as CD55, CD46, and CD59 that are expressed on the cell membrane of the host cell into which the virus is enveloped. For example, most of the anticancer vaccinia viruses produced are in the form of "intracellular mature viruses (IMVs)," which do not express complement regulatory proteins on the surface and whose activity is greatly reduced by complement. For this reason, research and development is needed to improve the anticancer activity of anticancer viruses when they are injected intravenously. [Detailed Description of the Invention] Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a novel anticancer virus, specifically, an anticancer virus in which the expression of the thymidine kinase (TK) gene is suppressed and which contains genes encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a complement regulatory protein.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which contains the anticancer virus as an active ingredient.

[0012] Another object of the present invention is to provide a gene construct for insertion into an anticancer virus, comprising all or part of a gene encoding a granulocyte-macrophage colony-stimulating factor, a gene encoding a cell membrane-spanning domain of an anticancer virus membrane protein, and a gene encoding a complement regulatory protein, operably linked to an early-late promoter and a late promoter for expression.

[0013] Another object of the present invention is to provide an anti-cancer adjuvant containing an anti-cancer virus as an active ingredient.

[0014] Another object of the present invention is to provide a method for preventing or treating cancer, which comprises the step of administering to an individual a composition containing the anti-cancer virus as an active ingredient.

[0015] Another object of the present invention is to provide a use of said anti-cancer virus or a composition containing the same for the prevention or treatment of cancer.

[0016] Another object of the present invention is to provide a use of said anti-cancer virus or a composition containing the same for the manufacture of a medicament for the prevention or treatment of cancer.

[0017] The technical problem to be achieved by the technical ideas of the invention disclosed in this specification is not limited to the problem of solving the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description. [Means for solving the problem]

[0018] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application belong to the scope of this application. Furthermore, the specific descriptions described below are not intended to limit the scope of this application.

[0019] In the present invention, in order to improve the anti-cancer activity of the above-mentioned anti-cancer virus when intravenously injected, the present inventors have made research efforts to develop a complement regulatory protein, for example, CD55, which has the highest activity, that can be expressed on the surface of the "intracellular mature virus" (IMV) to protect the anti-cancer virus from complement attack, and have completed the present invention.

[0020] As one embodiment for achieving the above-mentioned object of the present invention, the present invention provides an anti-cancer virus in which expression of the thymidine kinase (TK) gene is suppressed and which contains genes encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a complement regulatory protein.

[0021] In particular, in the present invention, as an example, the transmembrane domain in the membrane protein of the anticancer virus itself is linked to the CD55 gene so that the CD55 protein is expressed on the surface of the "intracellular mature virus" (IMV). This not only makes the virus resistant to the human complement system and maintains stable anticancer activity when administered intravenously, but also reduces the dosage of the anticancer virus, minimizing side effects from treatment.

[0022] The term "oncolytic vaccinia virus" of the present invention can be referred to as "oncolytic virus", which includes "recombinant viruses" engineered through the deletion of all or part of endogenous genes and the introduction of foreign genes. Such oncolytic viruses can be vaccinia virus, adenovirus, herpes simplex virus, retrovirus, reovirus, Newcastle disease virus, coxsackievirus, enterovirus, herpes virus, etc.

[0023] The term "thymidine kinase (TK)" as used herein refers to an enzyme involved in the biosynthesis of nucleotides. The thymidine kinase encoded by the TK gene can bind the phosphate at the gamma (γ) position of ATP to thymidine to produce nucleotides that constitute viral DNA. The TK can be a sequence such as, but is not limited to, GenBank: AAR17937.1 or AY313847.1. Specifically, the TK or its gene can be composed of, but is not limited to, the amino acid sequence of GenBank: AAR17937.1 or the nucleotide sequence of GenBank: AY313847.1. In addition, the TK or its gene can have a homology of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with the amino acid sequence of GenBank: AAR17937.1 or the nucleotide sequence of GenBank: AY313847.1.

[0024] The term "granulocyte-macrophage colony-stimulating factor (GM-CSF)" as used herein refers to a substance that helps produce more white blood cells, especially cells that will become granulocytes, macrophages, and platelets. It is a cytokine that belongs to the family of drugs called hematopoietic (blood-forming) agents, and is also known as sargramostim. GM-CSF is produced by many different cell types (e.g., activated T cells, B cells, macrophages, mast cells, endothelial cells, and fibroblasts) in response to cytokines or immune and inflammatory stimuli. Besides granulocyte-macrophage progenitors, GM-CSF is also a growth factor for erythrocyte, megakaryocyte, and eosinophil progenitors. For mature hematopoietic cells, GM-CSF is a survival factor for granulocytes, monocytes / macrophages, and eosinophils, activating their effector functions. GM-CSF has also been reported to have a functional role on non-hematopoietic cells: it can induce human endothelial cells to migrate and proliferate.

[0025] The GM-CSF may be a sequence such as, but not limited to, GenBank: M10663.1, etc. Specifically, the GM-CSF gene may be composed of or be a part of the nucleotide sequence of GenBank: M10663.1, and the GM-CSF gene may have about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the nucleotide sequence of GenBank: M10663.1.

[0026] The term "complement regulatory protein" as used herein refers to a protein that ingeniously regulates the complement activation pathway in vivo. Complement regulatory proteins are broadly classified into serotype (soluble) regulatory protein group and membrane-bound regulatory protein group. Serotype proteins include C4b-binding protein, factor H, SGP120, properdin (P), and membrane-type proteins include CRI (CD35), CR2 (CD21), CR3 (CD11b / CD18), CR4 (CD11c / CD18), DAF (CD55), membrane cofactor protein (MCP, CD46), and CD59. Among these, only P acts to enhance complement activity, while the others act to weaken it. The complement regulatory protein contained in the recombinant vaccinia virus of the present invention may be CD35, CD21, CD18, CD55, CD46, or CD59, and specifically may be CD55, but is not limited thereto. Specifically, the CD55 gene may be composed of or be a part of the nucleotide sequence of Genbank: NM_000574.3, and the CD55 gene may have about 90%, 91%, 92, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the nucleotide sequence of NCBI Reference Sequence: NM_000574.3. More specifically, the CD55 may be composed of the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.

[0027] As used herein, the term "transmembrane domain" is also called a transmembrane region or a membrane spanning region, and refers to a region of a membrane protein that penetrates and crosses the lipid bilayer.

[0028] The anticancer virus according to the present invention may further comprise a gene encoding a transmembrane domain of an anticancer virus membrane protein. Specifically, the vaccinia virus membrane protein may be D8L, A16L, F9L, G9R, H3L, L1R, A9L, A13L, A21L, A28L, E10R, G3L, H2R, I2L, J5L, L5R, or O3L, and may include all or part of the protein sequence, as shown in Table 1 below.

[0029] The complement regulatory protein of the present invention can be expressed independently of a gene encoding a transmembrane domain, or can be expressed in a linked fusion form.

[0030] [Table 1-1] [Table 1-2] [Table 1-3]

[0031] In this case, the cell membrane-spanning domain of the complement regulatory protein and the anticancer virus protein can be designed to include all or a part of the gene through a genetic recombination process. Specifically, the cell membrane-spanning domain of the vaccinia virus membrane protein of the anticancer virus can be composed of the amino acid sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:15, or the base sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16, but is not limited thereto.

[0032] The expression inhibition or gene inactivation of a gene according to the present invention means that a gene is not expressed or only a part of the gene is expressed, and the activity of the protein encoded by the gene is not expressed by deleting a part or the whole of the gene or inserting a foreign gene into the gene. The method of deleting the gene or the method of inserting a foreign gene can be performed by a method well known in the art. For example, the method of inserting a foreign gene disclosed in Methods described in Molecular Cloning, A Laboratory Manual, Second Edition, by J. Sambrook, EF Fritsch and T. Maniatis (2003), Cold Spring Harbor Laboratory Press, Virology Methods Manual, edited by Brian WJ Mahy and Hiliar O Kangro (1996) Academic Press and Expression of genes by Vaccinia virus vectors. Current Protocols in Molecular Biology, published by John Wiley and Son (1998), Chapter 16 can be used. Specifically, the expression of the thymidine kinase gene can be inhibited by inserting a foreign gene into a part or the whole of the thymidine kinase J2R region. More specifically, in one embodiment of the present invention, the T-Blunt TMThe foreign genes were inserted using PCR Cloning Kit (Solgent, Korea Cat No. SOT01-K020). The GM-CSF and complement regulatory protein contained in the anticancer virus of the present invention can be expressed under the control of the late-early VACV p7.5 promoter, vaccinia synthetic early-late promoter (pSEL), vaccinia synthetic late promoter (pSL), vaccinia modified H5 (mH5) promoter, vaccinia short synthetic early-late pS promoter, pLate promoter, pC11R promoter, pF11L promoter, psFJ1-10 synthetic early promoter, pHyb synthetic early promoter, any natural vaccinia early promoter, or late-early optimized (LEO) promoter, but are not limited thereto. In one example, the GM-CSF can be expressed under the control of the pSEL promoter, and the complement regulatory protein or CD55 can be expressed under the control of the pLate promoter. More specifically, the GM-CSF can be expressed under the control of the pSEL promoter of SEQ ID NO:3, and the complement regulatory protein can be expressed under the control of the pLate promoter of SEQ ID NO:4.

[0033] The anti-cancer virus of the present invention may further include a gene capable of enhancing the effect of cancer treatment. Such genes are not limited to, but may include, for example, anti-cancer therapeutic genes, various immune regulators, factors for decomposing structures such as intratumoral fibrous matter, etc., such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-17, IL-18, IL-21, IL-23, IL-24, interferon-α, interferon-β, interferon-γ, CCL3, CCL5, CXCR4, CXCL9, CXCL10, CXCL11, interleukin superagonist (IL-2 superagonist, IL-15 These may further include, but are not limited to, a superagonist, a TGF-β blockade, a TLR-2 agonist, a TLR-3 agonist, a TLR-7 agonist, a STAT-3 inhibitor, PTENα, p53, p63, p73, adenosine deaminase-2, a cancer-specific antigen, a cancer-associated antigen, hyaluronidase, collagenase, and protease.

[0034] In the present invention, anti-cancer viruses include vaccinia virus, adenovirus, herpes simplex virus, retrovirus, reovirus, Newcastle disease virus, coxsackievirus, enterovirus, herpes virus, etc., and as an example, the vaccinia virus may be, but is not limited to, Western Reserve (WR), NYVAC (New York Vaccinia Virus), Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, IHD-J (International Health Division-J), or IHD-W (International Health Division-White) strain.

[0035] In another aspect of the present invention to achieve the above object, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises the above anticancer virus as an active ingredient.

[0036] The term "active ingredient" as used herein means an ingredient that exhibits a desired activity alone or that can exhibit an activity together with a carrier that is inactive by itself.

[0037] The term "cancer (or tumor)" of the present invention includes all cancers, regardless of whether they are primary cancers or metastatic cancers. In the present invention, the cancer may be a solid cancer or a blood cancer. The solid cancer may be any of the following cancers: lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, kidney cancer, osteosarcoma, sarcoma, chondrosarcoma, and combinations thereof, but is not limited thereto. The blood cancer may be any of the following cancers: lymphoma, leukemia, multiple myeloma, and combinations thereof, but is not limited thereto.

[0038] The term "metastatic cancer" as used herein refers to cancer that occurs when cancer cells leave the primary organ, migrate to other organs, and grow there. The metastatic cancer may include, but is not limited to, both cases where cancer tissue grows from the primary cancer and directly invades surrounding organs, and cases where distant metastasis occurs along blood vessels or lymphatic vessels to other distant organs.

[0039] The term "prevention" as used herein means any action that suppresses or delays the onset of cancer through administration of the pharmaceutical composition of the present invention, and the term "treatment" as used herein means any action that beneficially changes or alleviates cancer by administration of the pharmaceutical composition of the present invention.

[0040] The specific dosage of the pharmaceutical composition containing the anti-cancer virus of the present invention can be selected by a person skilled in the art depending on factors such as the formulation method, the condition and weight of the patient, the sex and age of the patient, the degree of the disease, the drug form, the route and period of administration, the excretion rate, the reaction sensitivity, etc., and the dosage and frequency do not limit the scope of the present invention in any way. Specifically, the pharmaceutical composition of the present invention is 5 to about 10 13 It may include, but is not limited to, pfu (plaque forming unit) of an anti-cancer virus.

[0041] The term "pharmaceutical composition" of the present invention means a product manufactured for the purpose of preventing or treating a disease, and can be formulated into various forms according to a conventional method for use. For example, depending on the administration route, it can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated into external preparations and sterile injection solutions for use. Specifically, the administration route can be any suitable route, including topical, oral, intravenous, intramuscular, and direct absorption through mucosal tissue, and two or more routes can be used in combination. An example of a combination of two or more routes is when two or more drugs in a formulation according to the administration route are combined, for example, when one drug is administered primarily via the intravenous route and another drug is administered secondarily via the topical route.

[0042] The pharmaceutical compositions of the present invention may be for intratumoral, intravascular, intramuscular, or intraperitoneal administration, by way of example only, for intravenous or arterial administration.

[0043] The pharmaceutical composition of the present invention can be prepared in the form of a pharmaceutical composition for treating or preventing cancer, further comprising a suitable carrier, excipient, or diluent that is normally used in the preparation of a pharmaceutical composition, and the carrier can include a non-naturally occurring carrier. Specifically, the pharmaceutical composition can be formulated and used in the form of an oral preparation such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injection solution by a normal method. In the present invention, examples of the carrier, excipient, and diluent contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When preparing a formulation, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant that is commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stylate and talc are also used. Liquid preparations for oral administration include suspensions, liquid preparations, emulsions, syrups, etc., and include various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like can be used.As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc. can be used.

[0044] Specific formulations of pharmaceutical compositions are known in the art, and reference can be made, for example, to Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.

[0045] The pharmaceutical composition of the present invention can be administered to mammals such as mice, dogs, cats, cows, horses, pigs, and humans through various routes, preferably to humans. Any mode of administration can be envisaged, including, but not limited to, oral, intravenous, intraarterial, intramuscular, or subcutaneous injection.

[0046] For example, the anti-cancer virus of the present invention is resistant to the human body's complement system and maintains stable anti-cancer activity when administered intravenously, and also reduces the dose of the anti-cancer virus to maximize therapeutic efficacy, so that it can exhibit sufficient therapeutic efficacy even when administered intravenously.

[0047] In another embodiment for achieving the above object, the present invention provides a gene construct for insertion into vaccinia virus, the gene comprising all or part of a gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a gene encoding a transmembrane domain of a vaccinia virus membrane protein, and a gene encoding a complement regulatory protein, operably linked to an early-late promoter and a late promoter for expression, and a vector comprising the same.

[0048] In the present invention, the gene construct (or gene product) may be for insertion into the inactivated thymidine kinase gene region of an anticancer virus.

[0049] In the present invention, the inactivated thymidine kinase gene region includes a region in which the thymidine kinase gene is deleted in whole or in part.

[0050] In the present invention, the gene construct is located between the J1R and J3R regions in the anti-cancer virus.

[0051] In the present invention, the gene construct may include, but is not limited to, those exemplified in FIG. 1, and may further include various promoters and regulatory sequences for regulating gene expression in an operably linked form.

[0052] The terms "granulocyte-macrophage colony-stimulating factor (GM-CSF)", "transmembrane domain", and "complement regulatory protein" of the present invention are as defined above.

[0053] The gene construct of the present invention is intended for insertion into all or a part of the thymidine kinase of vaccinia virus, specifically, for insertion into all or a part of the thymidine kinase J2R region.

[0054] In another embodiment for achieving the above object, the present invention provides an anticancer adjuvant comprising the above recombinant vaccinia virus as an active ingredient.

[0055] The anticancer adjuvant means any form for enhancing the anticancer effect of anticancer drug or suppressing or improving the side effects of anticancer drug.The anticancer adjuvant of the present invention can be administered in combination with various kinds of anticancer drugs or anticancer adjuvant, and can show the same level of anticancer therapeutic effect even if the anticancer drug is administered at a lower dose than the usual dose of anticancer drug during the combined administration, so that it can perform safer anticancer treatment.

[0056] The administration route of the anti-cancer adjuvant can be any common route as long as it can reach the target tissue. The anti-cancer adjuvant of the present invention can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intrapulmonary, or intrarectally according to the purpose, but is not limited thereto. Furthermore, the anti-cancer adjuvant can be administered by any device that can transfer the active substance to the target cell.

[0057] The anti-cancer adjuvant of the present invention can be preferably formulated as an anti-cancer adjuvant by adding one or more pharma- ceutically acceptable carriers other than the active ingredient for administration.The carriers, excipients, or diluents contained in the anti-cancer adjuvant of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0058] The anticancer adjuvant of the present invention may be a formulation for parenteral administration, and the description of the formulation replaces the description of the formulation of the pharmaceutical composition.

[0059] Furthermore, any anticancer adjuvant disclosed in this technical field can be applied to the present invention without significant limitations.

[0060] In one embodiment of the present invention, the anti-cancer agent may be at least one selected from the group consisting of chemotherapeutic agents, biological therapeutic agents, radiation therapeutic agents, immunotherapeutic agents, hormonal therapeutic agents, antivascular therapeutic agents, cryotherapy agents, and toxin therapeutic agents, but is not limited thereto.

[0061] In another embodiment to achieve the above object, the present invention provides a method for preventing or treating cancer, comprising the step of administering to an individual a composition containing the recombinant vaccinia virus as an active ingredient.

[0062] The terms "cancer", "prevention" and "treatment" of the present invention are as defined above.

[0063] The individual may be a mammal, specifically, but not limited to, a human, cow, sheep, goat, horse, pig, dog, cat, rabbit, rat, mouse, fish, bird, etc.

[0064] The composition containing the recombinant vaccinia virus as an active ingredient can be appropriately administered by a skilled artisan according to the age, sex, weight, severity of symptoms, and administration route of the patient. The administration can be once a day or several times a day, and can be repeatedly administered at an appropriate interval.

[0065] The dosage of the composition containing the recombinant vaccinia virus as an active ingredient varies depending on the condition and body weight of the individual, the degree of the disease, the drug form, the administration route and the period, and can be appropriately selected by a person skilled in the art. 5 to about 10 13 The pfu (plaque forming units) can be administered and can include various values ​​or ranges within the above ranges.

[0066] In the method for treating cancer of the present invention, the composition may be administered via any common route as long as it can reach the target tissue. The composition of the present invention may be administered via routes such as oral administration and rectal administration, but is not limited thereto, and may also be administered via other routes depending on the purpose.

[0067] In the present invention, the cancer treatment method includes a treatment method in which the compound is administered in combination with a conventionally known anticancer agent and anticancer therapy.

[0068] To achieve the above object, another aspect of the present invention provides use of the above anticancer virus or a composition containing the same for the prevention or treatment of cancer.

[0069] To achieve the above object, another aspect of the present invention provides use of the above anticancer virus or a composition containing the same for the manufacture of a medicament for preventing or treating cancer. Effect of the Invention

[0070] The anticancer virus of the present invention maintains its efficacy even when administered intravenously, and can therefore be used to treat various solid carcinomas, blood cancers, and metastatic cancers in addition to superficial solid cancers. In addition, the anticancer virus of the present invention has resistance to the in vivo complement system by expressing a complement regulatory protein, and maintains stable anticancer activity, particularly when administered intravenously, thereby reducing the dosage of the virus and minimizing the side effects of anticancer drugs. Therefore, the anticancer virus of the present invention can be usefully used for the prevention or treatment of cancer. [Brief description of the drawings]

[0071] [Figure 1] FIG. 1 is a schematic diagram of a recombinant vaccinia virus, an anti-cancer virus of the present invention, in which the TK gene has been deleted and GM-CSF and CD55 fused to a transmembrane domain have been introduced. [Diagram 2] FIG. 2 shows the results of FACS examination to determine whether or not CD55 is expressed in osteosarcoma cells infected with the recombinant vaccinia virus of the present invention (SJ-v601 and SJ-v604 to SJ-v608). [Diagram 3] FIG. 3 shows the results of Western blotting to confirm whether the recombinant vaccinia viruses of the present invention (SJ-v601 and SJ-v604 to SJ-v608) express CD55. [Figure 4] FIG. 4 shows the results of confirming the survival rate of the recombinant vaccinia viruses of the present invention (SJ-v601 and SJ-v604 to SJ-v608) in 20% active human serum. [Diagram 5]FIG. 5 shows the results of confirming the recovery of the recombinant vaccinia viruses of the present invention (SJ-v601 and SJ-v604 to SJ-v608) using 20% ​​active human serum. [Figure 6] FIG. 6 shows the results of confirming the survival rates of JX-594 and SJ-v607 in the presence of 20% and 50% active human serum. [Figure 7] FIG. 7 shows the antitumor efficacy of JX-594 and SJ-v607 in an A549 lung cancer xenograft model confirmed by changes in tumor volume. [Figure 8] FIG. 8 shows the results of changes in body weight over time after administration of JX-594 and SJ-v607 to an A549 lung cancer xenograft model. [Figure 9] FIG. 9 shows the antitumor efficacy of JX-594 and SJ-v607 in a HCT-116 colon cancer xenograft model as determined by changes in tumor volume. [Figure 10] FIG. 10 shows the results of changes in body weight over time after administration of JX-594 and SJ-v607 to an HCT-116 colon cancer xenograft model. BEST MODE FOR CARRYING OUT THE INVENT

[0072] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0073] The present inventors prepared a vector into which human GM-CSF and human CD55 genes were inserted, and produced a recombinant vaccinia virus in which expression of the genes was induced by homologous recombination between the vector and vaccinia virus, and confirmed its characteristics as an anticancer substance.

[0074] Production Example 1: Production of recombinant vaccinia virus SJ-v601 lacking TK and expressing GM-CSF and CD55 As shown in Figure 1, the J2R region in the thymidine kinase domain was completely deleted in the Weiss strain of vaccinia virus, and CD55 and GM-CSF were inserted in this place. In order to express the CD55 in the viral envelope, a gene in which the GPI anchor region of hCD55 was deleted was fused with the transmembrane domain and membrane domain of the vaccinia virus membrane protein, and the promoter for expression was pLate. The specific process is as follows.

[0075] 1. Preparation of plasmid vectors expressing hGM-CSF and hCD55 genes To delete the J2R region of vaccinia virus, T-blunt TMThe J1R and J3R genes, which are the left and right flanking regions of the J2R region, were cloned into a vector (Solgent) using the NEBuilder (registered trademark) HiFi DNA Assembly Cloning Kit (NEW ENGLAND BioLabs, Catalog No. M5520A). The J1R and J3R DNAs were used by PCR amplification of the corresponding positions of JX-594 (Pexastimogene Devacirepvec, Pexa-Vec). The J1R is the same as the region of amino acids 1 to 154 of J1R (Protein id = AAR17936.1) of the vaccinia virus Acamins 2000 strain, but the double amino acid 118 is replaced from alanine to serine. J3R (Protein id = AAR17938.1) is the region of amino acids 1 to 145. The shuttle vector was completed by cloning hGM-CSF (GenBank: M10663.1) expressed by the vaccinia virus synthetic early-late promoter, the complement regulatory protein hCD55 gene (GenBank: NM_000574.3) expressed by the vaccinia virus late promoter, and the transmembrane domain region of vaccinia virus D8L into this vector. The gene sequence and amino acid sequence of hCD55 used are shown in SEQ ID NO: 1 and 2, respectively. The transmembrane domain regions of hGM-CSF and D8L were PCR amplified with JX-594, and hCD55 was PCR amplified with a vector cloned into pCMV3 plasmid (Sinobio, Catalog No. HG10101-UT). GM-CSF (GenBank: M10663.1), the region from amino acid 1 to 145, was expressed from a synthetic early-late promoter (aaaaattgaaattttttttttttttggaataaaata, SEQ ID NO: 3, pSEL, PCR amplified with Pexavec).hCD55 (GenBank: NM_000574.3) is a region of amino acids 1 to 352 with the GPI anchor region deleted, and the promoter for expression is a synthetic late promoter (ttttttttttttttttttttggcatataaata, SEQ ID NO: 4, pLate, synthesised by Macrogen). The 3' end of the hCD55 gene is linked to the transmembrane domain region of vaccinia virus D8L to allow CD55 to be expressed on the viral envelope membrane, and the information and base sequence of the transmembrane domain region of D8L used are shown in Tables 2 and 3.

[0076] [Table 2]

[0077] [Table 3]

[0078] 2. Production of SJ-v601 recombinant vaccinia virus by homologous recombination The SJ-v601 recombinant vaccinia virus was produced by homologous recombination between the wild-type vaccinia virus Wyeth strain and a previously constructed plasmid vector in which the expression of the hGM-CSF and hCD55 genes was induced.

[0079] The wild-type Wyeth strain was prepared by inserting the J2R region gene of the vaccinia virus Western Reserve (WR) strain (ATCC, Catalog No. VR-1354) into the inactivated J2R region (TK region) of JX-594 (Pexastimogene Devacirepvec, Pexa-Vec). J2R (Protein id = YP_232976.1) of the Western Reserve strain is the region located at amino acids 1 to 178. The J2R region gene of the Western Reserve strain was amplified by PCR, and the PCR product was homologously recombined with JX-594 to obtain the wild-type Wyeth strain.

[0080] To recombine SJ-v601, 143B osteosarcoma cells (Creative Bioarray) were infected with the wild-type Wyeth strain and transfected with a plasmid vector expressing hGM-CSF and hCD55. To select recombinant SJ-v601 plaques with deleted TK domain, 143B osteosarcoma cells were infected with vaccinia virus and transfected with the plasmid vector, and the cell lysates were treated with 5-Bromo-2'-deoxyuridine (BrdU), a TK deletion selection reagent, and cultured. The selected recombinant virus plaques were then subcultured twice more in 143B cells to obtain purified single clone SJ-v601. The nucleotide sequence of the recombination site of SJ-v601 was finally confirmed by sequencing.

[0081] Production Example 2: Production of recombinant vaccinia viruses SJ-v604 to SJ-v608 lacking TK and expressing hGM-CSF and CD55 The plasmid vector used for recombination was prepared in the same manner as in Example 1, except that the transmembrane domain of D8L was used, and SJ-v604 to SJ-v608 were prepared using the transmembrane domains of other membrane proteins. Information on the transmembrane domains used to prepare SJ-v604 to SJ-v608 is shown in the table below, and the transmembrane domain sequences of each membrane protein are also shown in the table below.

[0082] [Table 4]

[0083] [Table 5-1] [Table 5-2]

[0084] Finally, the structures of SJ-v601 and SJ-v604 to SJ-v608 prepared in Preparation Examples 1 and 2 are as shown in the table below.

[0085] [Table 6]

[0086] Example 1: Confirmation of CD55 expression in osteosarcoma cells of SJ-v601, SJ-v604 and SJ-v608

[0087] The recombinant vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 produced in Preparation Examples 1 and 2 were used to infect osteosarcoma cells, U-2 OS, and 16 hours later, FACS analysis was performed to confirm the presence or absence of CD55 expression. HeLa cells were used as a positive control, and JX-594 (Pexa-vec), which lacks TK and expresses GM-CSF and Lac-Z, was used as a negative control.

[0088] Specifically, U-2 OS were cultured at 4 × 10 per well in a 6-well plate. 5 The cells were seeded with vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 and cultured overnight. The next day, recombinant vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 were diluted to 0.5 plaque forming units (PFU) per cell in DMEM medium containing 2.5% heat-inactivated FBS. The diluted viruses were added to the U-2 OS cells inoculated the day before and infected at 37°C for 16 hours. After 16 hours, the infected cells were collected and washed with PBS, and then treated with Fixation / Permeabilization solution (BD, Catalog No. 565388) for cell fixation and permeabilization. Then, the cells were stained with anti-hCD55 antibody conjugated with APC fluorescent factor (BioLegend, Catalog No. 311312), and the expression of APC was measured by FACS (BD LSR Fortessa) and analyzed by Flowjo software.

[0089] As a result, as shown in FIG. 2, it was confirmed that CD55 was expressed in cells infected with SJ-v601 and SJ-v604 to SJ-v608 viruses.

[0090] In addition, purified intracellular mature virus (IMV) form of the virus was dissolved and Western blotting was performed, and as shown in Figure 3, it was confirmed that CD55 was expressed in the SJ-v601 and SJ-v604 to SJ-v608 viruses themselves.

[0091] Specifically, recombinant vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 were infected into HeLa cells for about 40 to 48 hours, respectively, and allowed to grow. The infected cells were centrifuged to remove the culture medium, and then a cell lysis buffer was added to lyse the cells. The cell lysate was treated with Benzonase to remove cell-derived DNA, and filtered through a cellulose acetate filter to remove cellular by-products. The filtrate was concentrated using a 36% sucrose cushion centrifugation method to prepare purified recombinant vaccinia viruses.

[0092] To isolate proteins present in the purified virus, the centrifuged virus precipitate was dissolved in RIPA lysis buffer (Thermo Scientific, Catalog No. 89900) and the amount of protein was quantified by BCA protein assay (Thermo Scientific, Catalog No. 23227). Proteins from each virus were diluted with loading buffer (Biosesang, Catalog No. S2002) to prepare 1ug per lane, and electrophoresed on SDS-PAGE gel (BIO-RAD, Catalog No. 4561083). The electrophoresed gel was transferred again to a nitrocellulose membrane (BIO-RAD, Catalog No. 1704270). For detection of the target proteins, the membrane was blocked with 5% skim milk (Difco, Catalog No. 232100) for 1 hour, and then treated with anti-hCD55 antibody (SantaCruz, Catalog No. sc-51733), anti-vaccinia virus A27 antibody (beiResources, Catalog No. NR-627), and anti-β-actin antibody (invitrogen, Catalog No. MA5-15739) overnight at 4°C. The next day, the membrane was washed 3-4 times with 1× TSBT, treated with secondary antibody conjugated with HRP, and then treated with hydrogen peroxide and luminol substrate, and chemiluminescence was confirmed with a detector.

[0093] Example 2: Confirmation of serum stability of SJ-v601, SJ-v604, and SJ-v608 in serum To confirm whether CD55 expressed on the viral envelope regulates complement in the blood, we measured its stability in serum as an in vitro proof-of-concept, by performing a plaque assay in which the virus was mixed with 20% active human serum containing viable complement functions.

[0094] Specifically, U-2 OS osteosarcoma cells were cultured in a 12-well tissue culture plate at 2.5 × 10 cells per well. 5 The 12-well plates were seeded with 12-well cells and cultured overnight. The next day, when the cells reached nearly 100% confluency, the recombinant vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 were diluted in serum-free DMEM medium to approximately 100 PFU per well. Human serum was added to the diluted viruses again at 20% of the total dose, and the diluted viruses were treated for 2 hours on plates in which U-2 OS cells were cultured. For the control group, serum-free DMEM medium was used instead of human serum. After 2 hours, the viruses were removed from the 12-well plates and treated with 1.5% carboxymethylcellulose solution. The 12-well plates were cultured at 37°C for 72 hours, and the formed plaques were stained with 0.1% crystal violet solution and counted. The ratio of the number of plaques formed by each recombinant virus cultured with 20% human serum is the same as in Figure 4, when the number of plaques formed without serum was taken as 100%. The same results are shown in FIG. 5 when the number of plaques produced by the control virus JX-594, which does not express hCD55, is set as 100%.

[0095] As a result, it was confirmed that the survival rate of the SJ-v600 series virus according to the present invention was higher than that of the control JX-594, as shown in Figures 4 and 5. For example, only 28% of plaques remained in JX-594, whereas SJ-v607, ​​which showed the highest survival rate, showed a survival rate of 76%.

[0096] Example 3: Confirmation of serum stability of SJ-v607 in serum To confirm whether CD55 expressed on the viral envelope regulates complement in the blood, we measured its stability in serum as an in vitro proof-of-concept. Specifically, we performed a plaque assay by mixing the virus with human serum (20%-50% active human serum) that has active complement function.

[0097] U-2 OS osteosarcoma cells were cultured in 12-well tissue culture plates at 2.5 × 10 cells per well. 5 The cells were seeded with 12-well plates and cultured overnight. The next day, when the cells reached nearly 100% confluency, recombinant vaccinia viruses SJ-v601 and SJ-v604 to SJ-v608 were diluted in serum-free DMEM medium to approximately 100 PFU per well. Human serum was added to the diluted viruses again at 20% to 50% of the total dose, and the diluted viruses were treated for 2 hours on plates cultured with U-2 OS cells. For the control group, 50% human serum in which the complement in the serum had been inactivated by heat inactivation was used. After 2 hours, the viruses were removed from the 12-well plates and treated with 1.5% carboxymethylcellulose solution. The 12-well plates were cultured at 37°C for 72 hours, and the formed plaques were stained with 0.1% crystal violet solution and counted. When the number of plaques formed in the 50% heat-inactivated human serum control group was taken as 100%, the percentage of the number of plaques formed by each recombinant virus cultured with 20% to 50% human serum was the same as in Figure 6.

[0098] The survival rates of the control group JX-594 and the recombinant vaccinia virus of the present invention, SJ-v607, ​​were measured in 20% and 50% serum, and it was confirmed that the survival rate of SJ-v607 was higher, and that the survival rate was higher in 20% serum than in 50% serum (Figure 6).

[0099] Example 4: Evaluation of antitumor effects in a lung cancer transplant model A549 human lung cancer cells were injected into one flank of NSG immunodeficient mice at 2 × 10 6 A xenograft mouse tumor model was established by subcutaneous injection of 1000 μg / mL ... 3 When the tumor growth inhibition rate reached 100%, SJ-v607 or JX-594 (control) was administered intravenously once to compare the tumor growth inhibition effects. SJ-v607 and JX-594 were administered at low concentrations (1 × 10 6 pfu) or high concentration (5 x 106 pfu).

[0100] The tumor size was measured in both the long and short axes using a caliper two to three times a week in all groups. The tumor size was calculated by dividing the short axis by two. The weight of the mice was also measured at each time point to evaluate the tumor size. 3 When this was reached, the experiment was terminated.

[0101] As a result, as shown in FIG. 7, when SJ-v607, ​​the recombinant vaccinia virus of the present invention, was administered, the tumor size was smaller than that of the control group JX-594, and the tumor size decreased after about 24 days (6 days after virus injection), unlike JX-594. 6 pfu, low concentration administration (1 × 10 6 pfu), and it was confirmed that the recombinant vaccinia virus of the present invention has a significantly superior antitumor effect. Furthermore, compared to the control group not administered with an anticancer drug, the anticancer virus administration caused a temporary weight loss, but this was quickly recovered, and the weight also remained within the normal range along with the reduction in tumor size (Figure 8).

[0102] Example 5: Evaluation of antitumor efficacy in a colon cancer xenograft model HCT-116 human colon cancer cells were inoculated into one flank of NSG immunodeficient mice at 5 × 10 5 A xenograft mouse tumor model was established by subcutaneous injection of 1000 μg / mL ... 3 When the 4-HT2048 antibody titer reached 1.0, SJ-v607 or the control JX-594 was added at 5 × 10 6 pfu was administered intravenously (single dose) to compare the efficacy in inhibiting tumor growth. The tumor size was measured in both the long and short axes of all groups 2-3 times a week using calipers. The tumor size was calculated by dividing the tumor size by (short axis x short axis x long axis). In addition, the weight of the mice was measured at each time point to evaluate the tumor size. 3When this was reached, the experiment was terminated.

[0103] As a result, as shown in Figure 9, when SJ-v607, ​​the recombinant vaccinia virus of the present invention, was administered, the tumor size was smaller throughout the entire period after administration compared to the control group JX-594, demonstrating a significantly superior antitumor effect. Furthermore, compared to the control group not administered with an anticancer drug, there was a temporary weight loss in the case of administration of the anticancer virus, but this was quickly recovered, and the weight remained within the normal range along with the decrease in tumor size (Figure 10).

Claims

1. An anti-cancer virus in which the expression of the thymidine kinase (TK) gene is suppressed and which contains genes encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and a complement regulatory protein, The anticancer virus further comprises a gene encoding a transmembrane domain of an anticancer virus membrane protein; a gene encoding a transmembrane domain of the anticancer viral membrane protein is fused to a gene encoding the complement regulatory protein; The anticancer virus is in the form of an intracellular mature virus (IMV), and the complement regulatory protein is expressed on the surface of the virus, The gene encoding the complement regulatory protein is a gene encoding CD55, the gene being composed of the sequence of SEQ ID NO: 1, the anti-cancer viral membrane protein is H3L; The anti-cancer virus is a vaccinia virus.

2. The expression of the thymidine kinase gene is suppressed by partial or total deletion of the gene or by insertion of a foreign gene into the gene. The anti-cancer virus according to claim 1.

3. The expression of the thymidine kinase gene is suppressed by inserting a foreign gene into a part or all of the region of the thymidine kinase gene. The anti-cancer virus according to claim 2.

4. The GM-CSF and complement regulatory proteins are expressed under the control of the late-early VACV p7.5 promoter, the vaccinia synthetic early-late promoter (pSEL), the vaccinia synthetic late promoter (pSL), the vaccinia modified H5 (mH5) promoter, the vaccinia short synthetic early-late pS promoter, the pLate promoter, the pC11R promoter, the pF11L promoter, the psFJ1-10 synthetic early promoter, the pHyb synthetic early promoter, any of the native vaccinia early promoters, or the late-early optimized (LEO) promoter, respectively; The anti-cancer virus according to claim 1.

5. the vaccinia virus is a Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, TashKent, Evans, International Health Division-J (IHD-J), or International Health Division-White (IHD-W) strain; The anti-cancer virus according to claim 1.

6. A pharmaceutical composition for preventing or treating cancer, comprising the anticancer virus according to any one of claims 1 to 5 as an active ingredient.

7. The cancer is a solid cancer or a hematological cancer. The pharmaceutical composition of claim 6.

8. The solid cancer is any one selected from the group consisting of lung cancer, colon cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymus cancer, stomach cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, kidney cancer, osteosarcoma, sarcoma, chondrosarcoma, and combinations thereof; The pharmaceutical composition of claim 7.

9. The blood cancer is any one selected from the group consisting of lymphoma, leukemia, multiple myeloma, and combinations thereof; The pharmaceutical composition of claim 7.

10. The composition is for intratumoral, intravascular, intramuscular, or intraperitoneal administration. The pharmaceutical composition of claim 6.

11. The composition is for intravenous or arterial administration. The pharmaceutical composition of claim 10.

12. A gene construct for insertion into an anti-cancer virus, comprising all or part of a gene encoding a granulocyte-macrophage colony-stimulating factor (GM-CSF), a gene encoding a transmembrane domain of a vaccinia virus membrane protein, and a gene encoding a complement regulatory protein, operably linked to an early-late promoter and a late promoter for expression, the early-late promoter is operably linked to all or a portion of a gene encoding GM-CSF, and the late promoter is operably linked to all or a portion of a gene encoding a complement regulatory protein; a gene encoding a transmembrane domain of the anticancer viral membrane protein is fused to a gene encoding the complement regulatory protein; The anti-cancer virus is in the form of an intracellular mature virus, and the complement regulatory protein is expressed on the surface of the virus; The gene encoding the complement regulatory protein is a gene encoding CD55, the gene being composed of the sequence of SEQ ID NO: 1, the anti-cancer viral membrane protein is H3L; The genetic construct, wherein the anti-cancer virus is a vaccinia virus.

13. The gene construct is for insertion into the inactivated thymidine kinase gene region of an anticancer virus. A genetic construct according to claim 12.

14. The gene construct is for insertion into the inactivated thymidine kinase gene region of an anticancer virus. A genetic construct according to claim 12.

15. An anti-cancer adjuvant comprising the anti-cancer virus according to any one of claims 1 to 5 as an active ingredient.

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